Methods for the quantitative determination of certain enzymes are based on the production of NADH (reduced nicotinamide adenine dinucleotide) or NADPH (reduced nicotinamide adenine dinucleotide phosphate) as a result of the enzymatic reaction on a substrate; the NADH or NADPH in turn reduces colorless 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (commonly known as INT) in the presence of an electron carrier such as phenazine methosulfate, to 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan (commonly known as INT formazan), which is bright red and can be measured spectrophotometrically. Concentrations of enzyme are determined by simultaneously running the test reaction on a reference sample containing known quantities of the enzyme and comparing spectrophotometric readings of the unknown against the reference sample.
Representative enzymatic determinations in which the above-described general method is used include the following: Babson, A. L. and Phillips, G. E., "A Rapid Colorimetric Assay for Serum Lactic Dehydrogenase," Clin. Chim. Acta 12, 210-215 (1965); Van Der Veen et al., "Isoenzymes of Creatine Phosphokinase in Tissue Extracts and in Normal and Pathological Sera," Clin. Chim. Acta 13, 312-316(1966); and Nachlas, M. M. et al., "The Determination of Lactic Dehydrogenase with a Tetrasodium Salt," Anal. Biochem., 1, 317-326 (1960).
The colored reference standard of this invention is useful not only in the determination of enzymatic activity but also for the determination of substrates or other products involving coupled enzymatic reactions yielding formazan. Thus, the colored reference standard of this invention may be used in any determination in which colorless 2-(para-iodophenyl)-3-(para-nitrophenyl)-5-phenyltetrazolium chloride is reduced to the bright red 1-(p-iodophenyl)-5-(p-nitrophenyl)-3-phenylformazan by means of enzymatic coupling reactions. For example, in the determination of glucose wherein INT formazan is produced via the hexokinase/glucose 6-phosphate dehydrogenase reaction; and in the diagnostic determination of such materials as adenosine triphosphate, glucose-6-phosphate, 6-phosphogluconate and the like, which involve similar reactions yielding INT formazan, the colored reference standard of this invention may be used to standardize the quantities of test product found in an unknown sample.
The accuracy of results using aforementioned methods for determining enzymatic activities depends, to a large extent, on the preparation and stability of the reference sample containing the known quantity of enzyme against which results with the test sample are compared. Furthermore, these enzymatic test methods involve double work, since in every instance, the test itself must be run on the reference sample, as well as on the unknown sample. such duplication requires extensive checking in order to insure that inaccuracies have not occurred.
Thus, the need for and the advantages of a stable colored reference standard for use in enzymatic determinations without simultaneous running of a control are obvious. However, such a product is not commercially available.